The spliceosome is an important emerging target for cancer therapy that has only recently been uncovered and remains to be significantly exploited. The development of highly selective anti-tumor agents continues to be a challenge in drug discovery and this fact has motivated researchers to search for new molecular targets that allow for the discovery of more selective anticancer agents than has been possible with compounds that inhibit many of the classic cancer targets. Recently, it was discovered that two unrelated natural products FR901464 (FR) and pladienolide (PD) both act by inhibition of the SF3b subunit of the spliceosome. Both of these natural products show activity in in vivo anti-tumor models. In particular, pladienolide and analogs have shown striking in vivo anti-tumor selectivity and efficacy, with a pronounced therapeutic window. In fact, a derivative of pladienolide B (E7107) has been advanced to Phase I human clinical trials. These natural products, and analogs derived from them, are quite complex and the synthesis of their analogs is quite demanding. It can readily be concluded that the discovery of more facile routes to active simplified compounds that work by modulation of the spliceosome is an important goal for the drug discovery community at large. We have recently reported the concise synthesis of novel highly stabilized synthetic analogs of FR that possess in vitro cytotoxicity IC50 values as low as 40-80 nM against multiple susceptible tumor lines and promising in vivo activity in a mouse tumor model (see Preliminary Results section). The overall long-term goal of this proposal is the development of a better understanding of spliceosome function, the development of new drugs for the treatment of human cancers that are most vulnerable to spliceosome modulation and elucidation of the mechanism of selective action of spliceosome modulators. We propose to develop both tool and optimized lead compounds capable of potent spliceosome modulation in vivo. We plan to accomplish this through exploration of the effects of our current active compounds on alternate splicing in tumors and through the refinement of our spliceosome modulators via multiple iterations of synthesis and in vitro testing of carefully designed new analogs, followed by several experimental cycles involving detailed investigations of the pharmacology of our lead compounds.